Antibody against illfdyfnr

ABSTRACT

Proteins useful in the diagnosis of proliferative disorders of the colon are present in nuclear matrix protein preparations and can be characterized by molecular weight, isoelectric point, and amino acid sequence. The proteins may be identified, for example, by 2D-gel electrophoresis or by specific binding partners, such as antibodies.

This application is a continuation of U.S. patent application Ser. No.10/350,367, filed Jan. 24, 2003 now abandonded, which claims the benefitof U.S. Provisional Patent Appl. No. 60/351,819, filed Jan. 25, 2002,and U.S. Provisional Patent Appl. No. 60/412,612, filed Sep. 19, 2002.

STATEMENT REGARDING GOVERNMENT SPONSORED RESEARCH

This invention was made with government support from the United StatesNational Institutes of Health. The government may have certain rights inthis invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to proteins associated with cellproliferative disorders of the colon including primary colon cancer andcolon cancer metastases to liver. The invention also relates to proteinsassociated with normal colon tissue.

Improvements in surgical procedures, adjuvant therapies, and screeningprograms have facilitated an overall decline in the mortality of coloncancer in the last 20 years (1). Colorectal cancer nevertheless remainsa significant disease accounting for 11% of all cancers in the UnitedStates with an estimated 130,200 new cases and 48,100 deaths expected inthe year 2001 (2). When diagnosed at an early localized stage, five-yearsurvival of colon cancer is 90%. Only 37% of incident cases arediagnosed at this stage, the remainder often presenting to a physicianwhen the tumor has become metastatic.

More effective screening and prevention measures for colorectal cancerare needed to address this public health problem. Early detectionprocedures for colorectal cancer have included tests for fecal blood oruse of endoscopy. The fecal blood test requires significant tumor size(sensitivity of 90% for small polyps and 75% for diminutive polyps) andhas a sensitivity of about 26%, which means 74% of patients withmalignant lesions will remain undetected (3). The fecal occult bloodtest fails to detect many early stage colon cancers because little ifany blood is released into the stool at that stage. Also the fecaloccult test is not very specific as a general screen, subjecting many toneedless discomfort and risk in subsequent full bowel examination.

Visualization of precancerous and cancerous lesions by endoscopy iseffective in early detection but is an invasive method with attendantsignificant risk of complications (4,5). For example, the cecum isreached in 80%–95% of procedures (22) and incomplete colonoscopiesrequire either a repeat colonoscopy or supplemental barium enema.

The complications and cost of the colonoscopy are considerable and theappropriate frequently at which this procedure should be used for cancerscreening tool is unknown. Furthermore, the procedural competence variesconsiderably among endoscopists. Thus, colonoscopy is presently notuseful for screening the general population for colon cancer.

Much effort over the years has been directed to the identification ofimproved diagnostic markers for colon cancer that enable reliable earlycancer detection or provide early prognostic information.Carcinoembryonic antigen (CEA), which is a tumor-associatedglycoprotein, was found to be expressed at increased levels in 95% ofcolorectal, gastric and pancreatic cancers, and in the majority ofbreast, lung and head and neck carcinomas (6). Diagnostic blood testsfor CEA are in use for following the course of therapy in the managementof colorectal cancer. In postoperative follow-up, CEA appears to be auseful marker of recurrence (sensitivity, 77%; specificity 98%), mainlyfor liver metastasis, but it has also been shown that only half ofcolorectal cancers shed CEA levels sufficient for their detection inmonitoring therapy (7,8).

The utility of CEA in detecting recurrences is controversial and has yetto be widely applied (9,10). Elevated CEA levels have been reported inpatients with nonmalignant disease and many patients with colon cancerhave normal CEA levels in the serum, especially during the early stageof the disease (7,11). In light of the currently available data, serumCEA determination possesses neither the sensitivity or specificityneeded as an early screening test for colorectal cancer in theasymptomatic population (12).

Changes in nuclear shape, size and DNA organization including majormorphological transformation are unique characteristics of cancer cellshas been used to diagnose cancer. Nuclear structure is determined by thescaffolding of the nucleus, the nuclear matrix. The nuclear matrixconsists of the peripheral lamins, protein complexes, an internalribonucleic protein network, and residual nucleoli (23). The nuclearframework consists of approximately 10% of the nuclear proteins, and isvirtually devoid of lipids, DNA, and histones (24). Most of the nuclearmatrix proteins identified to date are common to all cell types, butseveral identified NMPs are tissue and cell line specific and NMPs havebeen shown to undergo change with differentiation (25, 26).

Cell type-specific “fingerprinting” of aberrant nuclear matrix proteinsand their appearance in cancer development has led to the analysis ofnuclear matrix protein composition of a variety of tumors in an effortto determine whether these proteins can be developed as diagnosticand/or prognostic markers for cancer. By means of high resolution,two-dimensional electrophoresis, specific nuclear matrix proteinalterations have been demonstrated to exist in primary cancers of theprostate, bladder, renal and colon (27–30). The detection of nuclearmatrix proteins in the serum of patients with various types of cancerhas been accomplished (31).

SUMMARY OF THE INVENTION

In accordance with one aspect of the invention, there is provided apurified protein present in cancerous colon cells but absent or reducedin amount in normal colonic epithelial cells. The specified proteinsinclude, CC2, CC3, CC4, CC5, CC6a, CC6b, L1, L2 and L5. The apparentmolecular weight, isoelectric point and partial amino acid sequence forthese proteins are provided in the context of the examples herein. In apreferred embodiment the proteins are detected in nuclear matrixpreparations (“NMPs”).

In accordance with another aspect of the invention, proteins areprovided that are present in normal epithelial colonic cells but thatare absent or present in reduced amount in cancerous colon cells. Thespecified proteins include N1–N6, the apparent molecular weight, andisoelectric point of which are provided (see Examples, infra). In apreferred embodiment, the N proteins are detected in nuclear matrixpreparations.

In accordance with yet another aspect of the invention, there isprovided purified proteins in cancerous colon cells and liver metastasisderived therefrom which are useful as colon cancer diagnostic markers.The specified proteins include L1, L2, L3, L4 and L5, the apparentmolecular weight and isoelectric point of which are provided below. In apreferred embodiment, proteins L1–L5 are detected in NMPs.

In accordance with a further aspect of the invention, there is providedbinding partners specific for the invention proteins. The bindingpartners may be used, for example, for diagnostic or therapeuticpurposes. Methods of producing binding partners are provided. Thebinding partner preferably is a monoclonal or polyclonal antibody.

In accordance with yet a further aspect of the invention, a method isprovided for diagnosing in a patient a cell proliferative disorder ofthe colon, preferably colon cancer, by analyzing tissue, stool, or bodyfluid from the patient for the presence of at least one protein that ispresent in cancerous colonic cells but absent or reduced in amount innormal colonic epithelial cells, or that is present in normal colonicepithelial cells but absent or reduced in amount in cancerous coloniccells. Such diagnostic proteins include, CC2, CC3, CC4, CC5, CC6a, CC6b,L1, L2 and L5 and N1-N6. The proteins may be detected by any of avariety of means including biochemical means such as 2-D gelelectrophoresis, detection with a specific binding partner or bydetermining the level of encoding mRNA. In one embodiment, theproliferative disorder is colorectal adenocarcinoma while in anotherembodiment, the proliferative disorder is colonic adenoma.

In accordance with still yet a further aspect of the invention, there isprovided a method of diagnosing colon cancer in a patient comprisinganalyzing tissue, stool or body fluid from the patient for the presenceof calreticulin. Calreticulin may be detected by any of a variety ofmeans including biochemical means such as 2-D gel electrophoresis,detection with a specific binding partner or by determining the level ofencoding mRNA. In a preferred embodiment, calreticulin is detected inNMP preparations.

In accordance with another aspect of the invention, there is provided amethod of evaluating colonic adenomas for potential to become malignant.The method comprises analyzing the adenoma for the presence of at leastone protein that is present in cancerous colonic cells but absent orreduced in amount in normal colonic epithelial cells, or present innormal colonic epithelial cells but absent or reduced in amount incancerous colonic cells. The proteins are CC2, CC3, CC4, CC5, CC6a,CC6b, L1, L2 and L5 and N1–N6. In a preferred embodiment, the proteinsare CC3, CC4, and CC5. The proteins may be detected by any of a varietyof means including biochemical means such as 2-D gel electrophoresis,detection with a specific binding partner or by determining the level ofencoding mRNA.

In accordance with a further aspect of the invention, there is provideda method of diagnosing colon cancer to liver metastasis in a patient.The method comprises analyzing a sample of liver for the presence of atleast one protein, wherein the protein is any of L1–L5. The proteins maybe detected by any of a variety of means including biochemical meanssuch as 2-D gel electrophoresis, detection with a specific bindingpartner or by determining the level of encoding mRNA.

DETAILED DESCRIPTION OF THE INVENTION

As noted, a key aspect of the present invention is the inventor'sprovides a purified protein that is present in cancerous colon cells butabsent or reduced in amount in normal colonic epithelial cells. Theseproteins along with their apparent molecular weight (defined bySDS-PAGE) and isoelectric point are as follows:

-   a) CC2 having a molecular weight of about 56 kD and a pI of about    6.22;-   b) CC3 having a molecular weight of about 43 kD and a pI of about    6.3;-   c) CC4 having a molecular weight of about 43 kD and a pI of about    6.2;-   d) CC5 having a molecular weight of about 42 kD and a pI of about    6.2;-   e) CC6a having a molecular weight of about 20 kD and a pI of about    6.9;-   f) CC6b having a molecular weight of about 20 kD and a pI of about    6.8.-   g) L1 having a molecular weight of about 50 kD and a pI of about    6.01;-   h) L2 having a molecular weight of about 20 kD and a pI of about    5.73; and-   i) L5 having a molecular weight of about 19 kD and a pI of about    5.88.

These proteins also may be defined by amino acid sequence shown inTables 5, 6 and 9, and by presence in a NMP preparation. For example, inthe case of CC2, it is defined as comprising one or more proteins havinga molecular weight of about 56 kD and an isoelectric point of about6.22, wherein the protein comprises amino acid sequence from any one ormore of SEQ ID NOs: 14–17 and 23. Thus, CC2 can be defined by itsmolecular weight and pI, its presence in an NMP preparation, and by SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO:23, or any combination of these sequences.

Also provided is a purified protein that is present in normal epithelialcolonic cells but absent or reduced in amount in cancerous colon cells.These proteins along with their apparent molecular weight (defined bySDS-PAGE) and isoelectric point are as follows:

-   a) N1 having a molecular weight of about 40 kD and a pI of about    5.5;-   b) N2 having a molecular weight of about 30 kD and a pI of about    5.94;-   c) N3 having a molecular weight of about 30 kD and a pI of about    5.88;-   d) N4 having a molecular weight of about 30 kD and a pI of about    5.80;-   e) N5 having a molecular weight of about 30 kD and a pI of about    5.73; and-   f) N6 having a molecular weight of about 18 kD and a pI of about    6.6.    Proteins N1–N5 can be defined by their molecular weight and pI, and    presence in an NMP preparation.

Further provided are various purified proteins useful as diagnosticmarkers for colon cancer metastases to the liver. These proteins alongwith their apparent molecular weight (defined by SDS-PAGE) andisoelectric point are as follows:

-   a) L1 having a molecular weight of about 50 kD and a pI of about    6.01;-   b) L2 having a molecular weight of about 20 kD and a pI of about    5.73;-   c) L3 having a molecular weight of about 17 kD and a pI of about    6.09;-   d) L4 having a molecular weight of about 17 kD and a pI of about    6.00; and-   e) L5 having a molecular weight of about 19 kD and a pI of about    5.88.    L1–L5 are useful as diagnostic markers of colon cancer metastatic to    liver because these proteins are more detectable in samples of liver    that contain colon cancer than in samples of normal liver tissue.    These proteins may be defined by their molecular weight and pI,    their presence in an NMP prepration and by amino acid sequence as    shown in Table 9.

As used herein, the term “absent or reduced in amount in normal colonicepithelial cells” means that the protein is not detectable in normalcolonic epithelial cells or is detectable in such cells but at a lowerlevel than that for colorectal carcinoma cells. In being more detectablein colorectal carcinoma versus normal colonic epithelial cells, theprotein can be detected in 80% or more of cancer samples versus 50% orless for comparable non-cancer samples. Similarly, in connection withproteins that are markers of colon cancer to liver metastases, the termabsent or reduced in amount means that the proteins are more detectablein samples of liver that contain colon cancer than in samples of normalliver tissue. In being “more detectable, proteins L1–L5 are detectablein 80% of colon to liver metastases versus 50% or less for donor samplesof normal liver tissue.

The increased detectability of L1–L5 in metastatic colon to liver tissueversus normal liver tissue is believed to result from an increased levelof expression of these proteins in colon cancer cells versus normalliver cells including hepatocytes. “Normal liver tissue” includes“normal donor liver tissue,” which refers to liver tissue from a donorthat does not have metastatic liver cancer and “adjacent normal livertissue,” which refers to normal liver tissue from a liver with coloncancer metastasis. The term “adjacent” is used to identify the type ofliver from which this “normal” liver tissue is obtained (i.e. metastaticliver) rather than to indicate any degree of proximity between suchnormal tissue and the metastatic cancer.

The percentage of 2D gels in which L1–L5 were detected in samples ofliver containing colon cancer metastases and in samples containingnormal liver tissue is shown in Table 7, (Example 5). Although L3 and L4proteins were detected in normal adjacent liver and in normal donorliver, the amount of protein detected was qualitatively speaking lowerin amount that in the cancer metastatic tissue.

Protein identification by silver stained—high-resolution,two-dimensional gel electrophoresis analysis of nuclear matrixpreparations is a preferred method for determining whether the inventionproteins are more detectable in a particular tissue than in anothertissue. Other approaches are possible and include detection of theproteins in the tissue or in tissue extracts using a binding partnerspecific for the protein or by detecting RNA encoding the protein.

In this description, the phrase “nuclear matrix” refers to a3-dimensional filamentous protein network that is present in theinterphase nucleus. The NMPs of the protein network provide a frameworkto maintain the overall size and shape of the nucleus and acts astructural attachment site for the DNA loops during interphase.

An “NMP preparation” is a preparation from a biological source (e.g., acell, tissue or body fluid) that is enriched in nuclear matrix proteins(“NMPs”). “Enriched” means that at least some NMP are present at ahigher frequency in the NMP that they are in the natural state (e.g., incell, tissue, or biological fluid). NMP preparations may be prepared bywell known methods in the art such as detergent and urea extraction (seeGetzenberg et al. reference 29). An NMP preparation that is enriched inNMPs may contain proteins that are not part of the nuclear matrix

The present invention provides binding partners specific for theinvention proteins. The binding partners are useful to detect thepresence of the protein in a sample. The protein and its binding partnerrepresent a binding pair of molecules, which interact with each otherthrough any of a variety of molecular forces including, for example,ionic, covalent, hydrophobic, van der waals, and hydrogen bonding, sothat the pair have the property of binding specifically to each other.Specific binding means that the binding pair exhibit binding with eachother under conditions where they do not bind to another molecule.Examples of types of specific binding pairs are antigen-antibody,biotin-avidin, hormone-receptor, receptor-ligand, enzyme-substrate,IgG-protein A, and the like. A preferred binding partner for theproteins of the invention is an antibody, but other desirable bindingpartners may comprise a nucleic acid (e.g. natural or synthetic DNA,RNA, gDNA, cDNA, mRNA, tRNA, etc.), a lectin, an oligosaccharide, aglycoprotein, a drug candidates (from, for example, a random peptidelibrary, a natural products library, a lectin library, a combinatoriallibrary, an oligosaccharide library or a phage display library), ametabolite, a vitamin, a lipid, a steroid, a metal, and the like.

In the present context, an “antibody” is a protein that is made up ofone or more polypeptides, substantially encoded by immunoglobulin genesor fragments of such genes. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as a myriad of immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) is a tetramer composed of twoidentical pairs “light” chains (each about 25 kD) and two identicalpairs of “heavy” chains (each about 50–70 kD). The N-terminus of eachchain defines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms “variable lightchain” (VL) and “variable heavy chain” (VH) refer to the variableportions of the light and heavy chains, respectively. Theantigen-recognition site or ligand/substrate-binding site of animmunoglobulin molecule is formed by three highly divergent stretcheswithin the V regions of the heavy and light chains known as the“hypervariable regions,” which are interposed between more conservedflanking stretches known as “framework regions.” In an antibodymolecule, the three hypervariable regions of a light chain and the threehypervariable regions of a heavy chain are disposed relative to eachother in three dimensional space to form an antigen binding surface.This surface mediates recognition and binding of the target antigen orligand/substrate. The three hypervariable regions of each of the heavyand light chains are referred to as “complementarily determiningregions” or “CDRs” and are discussed, for example, by Kabat et al. (15).The portion of the antigen that interacts with the CDRs of the antibodyis referred to as an epitope.

Antibodies exist as intact immunoglobulins or as a number ofwell-characterized fragments, such as those produced by digestion withvarious peptidases and those that can be made by recombinant DNAtechnology. Antibody fragments include Fab′ monomer, Fab′2 dimer, Fvfragment, single chain Fv (“scFv”) fragment, and the like. See e.g.,Huston et al., (16). Antibody fragments also include antibody formshaving a truncated or deleted segment of the light and/or heavy chainconstant region.

Antibodies of the present invention may be monoclonal or polyclonal. Ingeneral polyclonal antibodies are present in the sera of animalsimmunized by one or more injections of the invention proteins orfragments thereof. In general, monoclonal antibodies are prepared byobtaining a source of B cells from a suitably immunized animal,immortalizing the B cells, cloning populations of B cells fromindividual immortalized cells, and selecting clones making an antibodyof interest. Methods for making polyclonal antibodies and monoclonalantibodies are well known in the art. For example, see Harlow and Lane(17).

Accordingly, a method is provided for producing polyclonal antibodiesthat differentiate primary colon cancer from normal colon and/or coloncancer liver metastases from normal liver tissue. The method comprisesimmunizing an animal with one or more of the invention proteins andrecovering the antibodies. There is further provided a method ofproducing a monoclonal antibody that differentiate primary colon cancerfrom normal colon and/or colon cancer liver metastases from normal livertissue. The method comprises immunizing an animal with one or more ofthe invention proteins, removing B cells from the immunized animal andimmortalizing and isolating the B cells that produce an antibodyspecific for the protein. As an alternative to immortalization, nucleicacid encoding antibody heavy and light chains may be obtained fromimmune cells of the immunized animal. The nucleic acid can then becloned into appropriate expression vectors and used to transform,transfect or otherwise transduce host cells so that the host cellsproduce antibody derived from the heavy and light chains present in Bcells of the immunized animal. Host cells producing antibodies ofinterest may be selected.

An immunogen suitable for eliciting specific antibodies to the inventionproteins can comprise a purified or a partially purified preparation ofthe protein. A purified protein of the invention is at least 20% pure,preferably at least 40% pure, more preferably at least 60% pure, evenmore preferably at least 80% pure and still even more preferably atleast 90% and most preferably at least about between 95 and 99% pure.

The terms “protein,” “polypeptide,” and “peptide” are usedinterchangeably in this description to denote a polymer of amino acidresidues. The category of “protein” includes proteins associated withother molecules, such as a glycoprotein, a proteoglycan, a lipoprotein,a nucleic acid, and combinations thereof. Unless otherwise specified,the terms “a,” “an” or “the” mean one or more. The proteins of theinvention also include conservatively substituted variants thereof.Minor modifications of the primary amino acid sequence may result inproteins that have substantially equivalent activity as compared to thenatural polypeptide described herein. Conservative substitutions insequence, which denote the replacement of an amino acid residue by astructurally similar residue, are preferred. Examples of conservativesubstitutions include the substitution of one hydrophobic residue suchas isoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acids, or glutamine forasparagine, and the like. Such modifications may be deliberate, as bysite-directed mutagenesis, or may be spontaneous, and can includedeletion of non-essential amino acids. Modification includes deletion ofone or more amino acids, which may be used to develop a smaller activemolecule that has broader utility. All of the polypeptides produced bythese modifications are included herein as long as the biologicalactivity of the native protein remains the same. The proteins of theinvention also include fragments of the protein which are useful, forexample, to generate binding partners for the protein.

An NMP preparation can be used as a source of partially purified proteinfor the immunogen. The invention proteins may be purified by any of anumber of well-known purification procedures, including precipitation,chromatography, electrophoresis, immunological separations involvingmonoclonal or polyclonal antibodies, and the like. For example, apurified protein preparation can be obtained by 2-D gel electrophoresisof an NMP followed by excising the protein spot and usingelectrophoresis to elute the protein from the gel (see Examples).Alternatively, the proteins or their fragments may be synthesized by thewell-known solid phase peptide synthesis methods described, for example,by Merrifield (13) or by Stewart and Young (14), based on proteinsequences.

Purified preparations of the invention proteins or fragments thereofalso may be prepared by recombinant expression using vectors and hostcells well known in the art and commercially available such as aredescribed in Goeddel et al. (18). Host cells include, for example,mammalian, bacterial, yeast, and the like. The host cell may betransformed with recombinant DNA by conventional techniques known in theart. For example, where the host is prokaryotic, such as E. coli, onecan prepare competent cells, which are capable of DNA uptake, from cellsharvested after the exponential growth phase and subsequently treated bythe CaCl₂ method, by conventional procedures. Transformation also can beperformed by forming a protoplast of the host cell or byelectroporation. A eukaryotic host may be transformed with foreign DNAusing well known procedures such as by calcium phosphatecoprecipitation, microinjection, electroporation, encasement inliposomes, virus vectors, and the like. Recombinant DNA also may beintroduced by transfection and transduction methods well known in theart.

Eukaryotic cells can also be co-transformed with DNA sequences encodingthe proteins of the invention, and a second foreign DNA moleculeencoding a selectable phenotype, such as the herpes simplex thymidinekinase gene. Another method is to use a eukaryotic viral vector, such assimian virus 40 (5V40) or bovine papilloma virus, to transiently infector transform eukaryotic cells and express the protein.

The invention proteins or fragments thereof may be expressed as a fusionto a foreign polypeptide, for example, a bacterial ligand bindingsequence such as GST or Staphylococcal Protein A. Fusion to GST, forexample, provides an efficient means to purify the fusion product orincrease its immunogenicity (see, e.g., Uhlen and Moks, (19)).Immunogenicity may also be enhanced by chemically coupling the proteinor fragment to a suitable immunogenic carrier protein. Carrier proteinsuseful for the present invention have molecular weights of at leastabout 20,000 Daltons. Carrier proteins useful in the present inventioninclude, for example, GST, hemocyanins such as from the keyhole limpet,serum albumin or cationized serum albumin, thyroglobulin, ovalbumin,various toxoid proteins such a tetanus toxoid or diphtheria toxoid,immunoglobulins or heat shock proteins. Methods to chemically couple apolypeptide to a carrier protein are well known in the art and includeconjugation by a water soluble carbodiimide such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, conjugationby a homobifunctional cross-linker having, for example, NHS ester groupsor sulfo-NHS ester analogs, conjugation by a heterobifunctionalcross-linker having, for example, and NHS ester and a maleimide groupsuch as sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, conjugation with gluteraldehyde, and thelike. For example, see Hermanson, (20) and U.S. Pat. No. 4,608,251 andNo. 4,161,519.

Binding partners such as polyclonal or monoclonal antibodies can be usedto detect the invention proteins in immunoassays such as RIA, EIA, andthe like. Such assays may be competitive or non-competitive and maybased on a direct or an indirect format. The binding partner can be usedin liquid phase and/or bound to a solid-phase carrier. Carriers includeglass, polystyrene, polypropylene, polyethylene, dextran, nylon, naturaland modified cellulose, polyacrylamide, agarose, magnetite, and thelike. The nature of the carrier can be either soluble or insoluble. Asis typical of immunoassays, the binding partner or the invention proteinmay be detectably labeled in any of various ways well known in the art.Binding partners also may be used to detect the invention proteins inelectrophoretically dispersed gels (e.g., a 2-D gel) or attached to asolid phase membrane such as a Western blot.

An immunogen comprising the invention proteins or fragments thereof mayalso be administered with an adjuvant either by mixing with the proteinor fragment or by conjugating or otherwise linking to the adjuvant. Avariety of adjuvants are known including Freund's (complete andincomplete), alum, muramyl dipeptide, BCG, LPS, Ribi Adjuvant System®,TiterMax®, and the like. One skilled in the art would know which type ofadjuvant is appropriate to use in a given circumstance.

The present invention also provides a method of diagnosing aproliferative disorder of the colon in a patient, the method comprising:analyzing tissue, stool or body fluid from the patient for the presenceof at least one protein that is present in cancerous colonic cells butabsent or reduced in amount in normal colonic epithelial cells, orpresent in normal colonic epithelial cells but absent or reduced inamount in cancerous colonic cells, wherein the protein is selected fromthe group consisting of:

-   a) CC2 having a molecular weight of about 56 kD and a pI of about    6.22;-   b) CC3 having a molecular weight of about 43 kD and a pI of about    6.3;-   c) CC4 having a molecular weight of about 43 kD and a pI of about    6.2;-   d) CC5 having a molecular weight of about 42 kD and a pI of about    6.2;-   e) CC6a having a molecular weight of about 20 kD and a pI of about    6.9;-   f) CC6b having a molecular weight of about 20 kD and a pI of about    6.8.-   g) L1 having a molecular weight of about 50 kD and a pI of about    6.01;-   h) L2 having a molecular weight of about 20 kD and a pI of about    5.73;-   i) L5 having a molecular weight of about 19 kD and a pI of about    5.88;-   j) N1 having a molecular weight of about 40 kD and a pI of about    5.5;-   k) N2 having a molecular weight of about 30 kD and a pI of about    5.94;-   l) N3 having a molecular weight of about 30 kD and a pI of about    5.88;-   m) N4 having a molecular weight of about 30 kD and a pI of about    5.80;-   n) N5 having a molecular weight of about 30 kD and a pI of about    5.73; and-   o) N6 having a molecular weight of about 18 kD and a pI of about    6.6.

As already described, the protein may be detected by any of a variety ofmeans including biochemical means such as 2-D gel electrophoresis,detection with a specific binding partner such as a monoclonal orpolyclonal antibody, and the like. The protein also may be detected bydetermining the level of mRNA encoding the protein through hybridizationwith an appropriate oligonucleotide probe. A “patient” in this regard isa mammal, preferably a human.

In this description, the phrase “proliferative disorder of the colon”denotes malignant as well as non-malignant (or benign) disorders of thecolon, including but not limited to the colonic epithelium. Suchdisorders include polyps such as with atypia or dysplasia, sessilevillous adenomas, pedunculated tubular adenomas, and the like. The cellscomprising these proliferative disorders often appear morphologicallyand genotypically to differ from the surrounding normal tissue. Theproliferative disorder may be associated, for example, with expressionof the CC or L proteins of the invention, in the latter case,particularly L1, L2 and L5. Expression of a protein identified herein atan inappropriate time during the cell cycle or in an incorrect cell typemay result in a cell-proliferative disorder. The protein-encodingpolynucleotide in the form of an antisense polynucleotide (or ribozyme)is useful in treating hyperplasia and malignancies of the colon. In apreferred embodiment, the proliferative disorder is colon cancer. Table2 (Example 2) shows the percentage of 2-D gels in which L1, L2 and L5were detected in nuclear matrix preparations of normal adjacent colonictissue and normal donor colonic tissue.

Also provided herein is a method of evaluating colonic adenomas forpotential to become malignant, the method comprising: analyzing theadenoma for the presence of at least one protein that is present incancerous colonic cells but absent or reduced in amount in normalcolonic epithelial cells, or present in normal colonic epithelial cellsbut absent or reduced in amount in cancerous colonic cells, wherein theprotein is selected from the group consisting of:

-   a) CC2 having a molecular weight of about 56 kD and a pI of about    6.22;-   b) CC3 having a molecular weight of about 43 kD and a pI of about    6.3;-   c) CC4 having a molecular weight of about 43 kD and a pI of about    6.2;-   d) CC5 having a molecular weight of about 42 kD and a pI of about    6.2;-   e) CC6a having a molecular weight of about 20 kD and a pI of about    6.9;-   f) CC6b having a molecular weight of about 20 kD and a pI of about    6.8.-   g) L1 having a molecular weight of about 50 kD and a pI of about    6.01;-   h) L2 having a molecular weight of about 20 kD and a pI of about    5.73;-   i) L5 having a molecular weight of about 19 kD and a pI of about    5.88;-   j) N1 having a molecular weight of about 40 kD and a pI of about    5.5;-   k) N2 having a molecular weight of about 30 kD and a pI of about    5.94;-   l) N3 having a molecular weight of about 30 kD and a pI of about    5.88;-   m) N4 having a molecular weight of about 30 kD and a pI of about    5.80;-   n) N5 having a molecular weight of about 30 kD and a pI of about    5.73; and-   o) N6 having a molecular weight of about 18 kD and a pI of about    6.6; wherein detection of said proteins indicates an increased    potential to become malignant. In a preferred embodiment, the    proteins are CC3, CC4 and CC5.

The invention also contemplates the diagnosing of colon cancer to livermetastasis in a patient, by analyzing tissue or body fluid from thepatient for the presence of at least one protein selected from the groupconsisting of:

-   a) L1 having a molecular weight of about 50 kD and a pI of about    6.01;-   b) L2 having a molecular weight of about 20 kD and a pI of about    5.73;-   c) L3 having a molecular weight of about 17 kD and a pI of about    6.09;-   d) L4 having a molecular weight of about 17 kD and a pI of about    6.00; and-   e) L5 having a molecular weight of about 19 kD and a pI of about    5.88.

Detection of proteins according to the present invention can allowdifferentiation among colonic cell proliferative disorders that have apotential to become malignant or to metastasize, respectively. Theprotein compositions described herein also are useful as markers forearly diagnosis of a colonic cell proliferative disorder such ascolorectal cancer and the early detection of recurrence, and itsmetastastic diseases derived therefrom, knowledge that is central to theeffective treatment of this disease. In addition to their useindividually, detection of any two or more invention proteins can becombined to diagnose proliferative disorders of the colon includingcolon cancer and its liver metastases. Detection of the inventionproteins also may be combined with detecting other colon cancer markers,such as CEA or previously reported colon cancer NMPs (32), to diagnosethe colon cancer and its liver metastases.

The particular proteins or fragments described herein may be detectedindirectly, for example, by detecting the level of encoding mRNA. Thus,the present invention provides a purified polynucleotide sequenceencoding the above-identified protein or fragments of the precedingembodiments. Also provided is a nucleic acid probe that hybridizes tothe polynucleotide sequence encoding the above-mentioned protein orfragments thereof (or by hybridization to the complementary sequence).The term “nucleic acid” as used herein refers to a deoxyribonucleotideor ribonucleotide polymer in either single—or double-stranded form, andalso encompasses known analogs of natural nucleotides that can functionin a similar manner as naturally occurring nucleotides. The probe ispreferably single stranded and preferably comprises at least about 14nucleotides in length, more preferably at least about 18 nucleotides inlength, and most preferably about 25 nucleotides in length. The nucleicacid probe can be detectably labeled such as with a radioisotope, abioluminescent compound, a chemiluminescent compound, a fluorescentcompound, a metal chelate, an enzyme, and the like.

Also provided is a kit for detecting a colonic cell proliferativedisorder. The kit may include a binding partner or nucleic acid probethat is specific for any one or more of the invention proteins. Thebinding partner or probe can be labeled for ease of detection. The kitmay have reagents in different vials and may include positive andnegative controls, buffers such as for conducting the reactions, and adirectional insert. The kit also may have an oligonucleotide primer thatpermits amplification of a target polynucleotide sequence encoding oneof the invention proteins, for example, by polymerase chain reaction(PCR) amplification.

The present invention also provides methods to treat colon cancer andits liver metastases, by reducing the level of expression or activity ofa protein which is present primary colon cancer cells and livermetastases derived therefrom. Such proteins include any of the CC or Lproteins disclosed herein. The level of expression of the protein can bereduced, for example, by treating the individual with an antisensenucleic acid or a ribozyme.

An antisense nucleic acid is a short DNA or RNA oligonucleotide designedto be complementary to a specific gene sequence. The purpose in usingantisense to alter specific gene expression resulting from the bindingof the antisense oligonucleotide to the gene sequence. Antisensemolecules that bind to a specific region of the DNA helix may result intriplex formation since a third strand is formed at the site of bindingto the two stands comprising the helix. Antisense oligonucleotides thattarget RNA may be either non-catalytic or catalytic (i.e., ribozymes).Binding of non-catalytic antisense molecules to the target RNA can blockfurther RNA processing or transport through any of several possiblemechanisms, including, for example: 1) transient inhibition by theprevention of ribosome binding to the RNA by masking its binding site onthe mRNA; 2) permanent inhibition of the process by which RNase Hdegrades RNA in RNA/DNAs hybrids, an enzyme present in most cells; and3) permanent inhibition by cross-linking the oligonucleotide to thetarget RNA.

The antisense compounds preferably comprise between about 8 to about 30nucleobases (i.e., from about 8 to about 30 linked nucleosides), andmore preferably from about 12 to about 25 nucleobases. The antisenseoligonucleotide can be linear or circular in configuration. Antisensecompounds useful herein may include oligonucleotides containing modifiedbackbones or non-natural internucleoside linkages. Preferred modifiedoligonucleotide backbones include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3-5′ to 5′-3′ or 2′–5′ to 5′-2′. Extensivecitation to methods of preparing antisense oligonucleotides can befound, for example, in U.S. Pat. No. 6,210,892 to Bennett.

Where the cell proliferative disorder is characterized as havingunder-expression of an invention protein (e.g., N1 or N6) apolynucleotide sequence encoding the missing under-expressed protein(such as for a growth or tumor suppressor protein) can be administeredto increase the level of expression. This can be achieved by introducinginto the cells of a host subject an expression vector comprising apolynucleotide sequence encoding any one or more of the inventionproteins. Preferably, the expression vector is introduced into the cellsof the host subject ex vivo, yielding transformed cells, and thetransformed cells are then reintroduced into the subject. This can beachieved, for example, with an RNA virus vector such as a retroviralvector. Cells transformed to express the proteins are also useful, forexample, as a tumor vaccine. Methods of preparing vectors for deliveringsense or antisense nucleic acid into cells (in vivo or in vitro) andmethods of formulating these delivery vehicles for administration arewell known in the art. See, e.g., U.S. Pat. No. 5,824,490 to Coffey etal.

The level of expression of the invention protein also can be reduced bytreating the individual with a compound that effects a regulatory regioncontrolling expression such a promoter or an enhancer or that modulatesthe activity of the proteins. Such compounds may be identified by a)incubating the proliferative cells (e.g., colorectal cancer cells)expressing the invention protein with a test composition underconditions that allow the cells and test composition to interact, and(b) measuring whether the test composition blocks or enhances thefunction of the protein. Caco-2 cells are an example of a colorectalcancer cell line that is useful in this regard.

The invention will be described in greater detail by reference to thefollowing non-limiting examples.

EXAMPLES Example 1 Methods for Isolation and Purification of NMPs

A. Tissue Processing:

Colon adenocarcinoma liver, metastases samples (N=12) and matchedadjacent normal liver tissues (N=12) were collected through the EarlyDetection Research Network (EDRN) of the University of PittsburghMedical Center under institutional IRB approval. The patients ranged inage from 44–75 with a mean age of 62.4 years. Sixty percent of thesample population was female. The carcinomas, staged according thestandard TNM system, were categorized as shown in Table 1.

Colon adenocarcinoma samples (N=10) and matched adjacent normal livertissues (N=10) also were collected through the Early Detection ResearchNetwork (EDRN) of the University of Pittsburgh Medical Center underinstitutional IRB approval. The patients ranged in age from 36–82 with amean age of 71 years. Sixty percent of the sample population was female.Staging for these carcinomas categorized as shown in Table 1.

TABLE 1 Stage and UI Grade of Colon Carcinoma Tumors Used for NMPPreparation Clinical Parameter Result (number of samples) Tumor locationRight hemicolon (n = 5) Left heimcolon (n = 5) Tumor Stage (UICC) I:T_(1–2)N₀M₀ (n = 0) II: T_(3–4) N₀M₀ (n = 5) III: T_(1–4)N_(1–2)M₀ (n =4) IV: T_(1–4)N_(1–2)M₁ (n = 1) Tumor Grade G1 (n = 0) G2 (n = 9) G3 (n= 1)

Normal liver tissue was obtained from trauma victims including onesample from a gunshot victim and two from an automobile accident victim(N=3). The patients ranged in age from 36–48 with a mean age of 40.3years. Two of these normal individuals were male and one female. None ofthe patients had liver cirrhosis. Thirty percent of the patients hadmild steatosis. Diagnosis was obtained from pathology reports, whichaccompanied each specimen and was confirmed histologically. Tissues werestored at −80° C. prior to processing. Sporadic colon adenocarcinomasamples and matched adjacent normal tissues and normal donor colontissues were collected as previously described (30).

Normal colon tissue was obtained from trauma victims; Two of theseindividuals had gunshot wounds, one suffered from automobile trauma andone was an organ donor (N=4). The patients ranged in age from 20–59 witha mean age of 47.2 years. The normal colon donors were all male.Diagnosis was obtained from pathology reports, which accompanied eachspecimen and was confirmed histologically.

The colon cancer cell lines, SW480 and Caco-2, were obtained from theAmerican Type Culture Collection (Manassas, Va.). Both cell lines wereestablished from primary human colon cancer cells. The SW480 cell linewas grown in Leibovitz medium with 10% fetal bovine serum at 37° C.without CO₂. The Caco-2 cell line was grown in Dulbecco's minimalessential medium (DMEM) supplemented with 10% fetal bovine serum, 1%L-glutamine (200 mM), 1% penicillin/streptomycin, 1% sodium pyruvate(100 mM), 1% MEN non-essential amino acids, 1.5% HEPES Buffer (1M) at37° C. in a 5% CO₂ atmosphere.

The colon cancer cell line CX-1 was a kind gift from Lee Y J Ph.D.University of Pittsburgh. The cell line has been established fromprimary human colon cancer cells. The cell line was grown in RPMI-1640media with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C. in a 5% CO₂ atmosphere.

Normal human hepatocytes (50×10⁶), obtained from a 63 year-old femaleorgan donor, were a gift from Dr. Stephen Strom (University ofPittsburgh, Pa). Human primary liver cancer cell lines, huh 7, HepG 2,were a kind of gift of Dr. George Michalopoulos, University ofPittsburg, Pa. Both cell lines were grown in DMEM with 10% fetal bovineserum and 1% penicillin/streptomycin at 37° C. in a 5% CO₂ atmosphere.

B. Nuclear Matrix Preparation:

Nuclear matrix proteins were extracted from various cells and tissuesaccording to the method of Getzenberg et al (29). In brief, tissues werefinely minced into small pieces and homogenized with a Teflon® pestle onice with 0.5% Triton X-100 in a solution containing 2 mM vanadylribonucleoside (Rnase inhibitor) to release the lipids and solubleproteins. The homogenate was filtered through a 350 μm nylon mesh.Treatment with DNAse and RNAse was performed to remove the solublechromatin, resulting in a remaining fraction comprising intermediatefilaments and NMPs. This fraction then was disassembled with 8 M urea,and the insoluble components consisting of carbohydrates andextracellular matrix were removed by centrifugation. After dialysis toremove the urea, intermediate filaments were allowed to reassemble andwere subsequently removed by centrifligation.

The NMPs then were precipitated with ethanol and resuspended in 2Dsample buffer consisting 9 M urea, 65 nM3-((3-cholamidopropyl)-dimethyl-ammonio)-1-propane-sulfonate, 2.2%amhpolytes and 140 mM DTT, and quantitated by Coomassie Plus proteinassay (Pierce Chemical Co., Rockford, Ill.) with bovine serum albumin asa standard. The pellet following ethanol precipitation containing NMPsrepresented <1% of the total starting cellular protein.

C. High Resolution, Two-dimensional Electrophoresis:

This procedure was performed using the Investigator 2-D gel system(Genomic Solution, Ann Arbor, Mich.) as described previously (29,33–34). One hundred μg of protein were loaded per gel onto a capillarysize IEF column. One dimensional isoelectric focusing was carried outfor 18,000 volt-hours using 1 mm×18 inch tube gels after 1.5 hours ofprefocusing. The tube gels were extruded and placed on top of 1 mm SDSDuracryl (Genomic Solution, Ann Arbor, Mich.) high tensile strength PAGEslab gels. The gels were electrophoresed at 12° C. constant temperaturefor 4.5 to 5 hours. Gels were fixed with 50% methanol and 10% aceticacid. After thorough rinsing and rehydration, gels were treated with 5%glutaraldehyde and 5 mM DTT after buffering with 50 mM Phosphate (pH7.2). The gels were stained with silver stain using the method of Wrayet al (Accurate Chemical Co., Westbury, N.Y.) (21). Molecular weights ofcolon NMPs were identified using standards provided by GenomicSolutions. Isoelectric points (PI's) were determined using carbamylatedstandards; BDH-distributed by Gallard-Schlessinger (Carle Place, N.Y.)and Sigma Chemical Co. (St. Louis, Mo.).

Multiple gels were run for each sample and multiple samples were run atdifferent times. The gels were analyzed using the BioImage 2DElectrophoresis Analysis System (BioImage, Ann Arbor, Mich.), thatmatches protein spots between gels and sorts the gels and protein spotsinto a database. Only protein spots clearly and reproducibly identicalin all gels of a sample type were taken into account as thoserepresenting the described NMP's.

Example 2 Identification of NMPs Characteristic of Colon Carcinoma orNormal Colonic Epithelium

A. Analysis of NMPs from Colorectal Cancer and Normal Colonic Epithelium

Evaluation of NMPs separated by high-resolution two-dimensional gelelectrophoresis identified seven NMPs of clinical value that wereexpressed primarily in colorectal adenocarcinomas were identified anddesignated as shown in Table 2. CC2 to CC6a,b were strongly expressed inall colon tumors, but were undetectable in adjacent normal tissue. Incontrast, CC1 was expressed in carcinoma extracts and adjacent normal ordonor tissue but the signal was stronger for colon cancers than eithersource of normal tissue.

Six NMPs of clinical value that were expressed primarily in normalcolonic epithelium were designated as shown in Table 2. N2–N6 wereexpressed in a minority of the colon cancers (about 20%), but wereexpressed in all adjacent normal and donor extracts. In contrast, N1 wasexpressed in most cancer extracts (about 70%) and in all normalextracts, but was more strongly but the signal was stronger for thenormal extracts. The characteristics of the identified colon carcinomaNMPs are summarized in Table 2.

TABLE 2 Characteristics of Colorectal Adenocarcinoma and Normal ColonicEpithelial NMPs Marker Molecular No. Weight (kD) PI Tissue ExpressionCC1 59 4.40 Cancer and normal epithelium but stronger for cancer (10/10cancer; 10/10 normal adjacent; and 4/4 normal donor) CC2 56 6.22 Canceronly (8/10 cancer; 0/10 adjacent normal; and 0/4 donor normal) CC3 436.27 Cancer only (10/10 cancer; 0/10 adjacent normal; and 0/4 donornormal) CC4 43 6.22 Cancer only (10/10 cancer; 0/10 adjacent normal; and0/4 donor normal) CC5 42 6.25 Cancer only (10/10 cancer; 0/10 adjacentnormal; and 0/4 donor normal) CC6a 20 6.86 Cancer and donor epitheliumbut not adjacent normal epithelium (8/10 cancer; 0/10 adjacent normal;and 4/4 donor normal) CC6b 20 6.81 Cancer and donor epithelium but notadjacent normal epithelium. (8/10 cancer; 0/10 adjacent normal; and 4/4donor normal) N1 40 5.50 Normal epithelium (adjacent and donor) andcancer (about 70% of samples) but stronger for normal (7/10 cancer;10/10 adjacent normal; and 4/4 donor normal) N2 30 5.94 Normalepithelium mainly** (2/10 cancer; 10/10 adjacent normal; and 4/4 donornormal) N3 30 5.88 Normal epithelium mainly (2/10 cancer; 10/10 adjacentnormal; and 4/4 donor normal) N4 30 5.80 Normal epithelium mainly (2/10cancer; 10/10 adjacent normal; and 4/4 donor normal) N5 30 5.73 Normalepithelium mainly (2/10 cancer; 10/10 adjacent normal; and 4/4 donornormal) N6 18 6.58 Normal epithelium mainly (1/10 cancer; 10/10 adjacentnormal; and 4/4 donor normal) *NC means that the reference describesnothing close in size or pI. **Refers to an expression frequency ofabout 10–20% for cancer samples.B. Analysis of CC NMPs from Colorectal Tumor Cell Lines

NMP preparations were isolated from two human colon cancer cell lines,Caco-2 and SW480, essentially as described for NMP preparation fromhuman tissue. These NMPs were analyzed by 2 dimensional gelelectrophoresis as described above for human tissue NMPs. Both celllines expressed none of the proteins, which are expressed in theadjacent or donor gels (N1–N6). The cell line SW480 expressed the coloncancer associated proteins, CC1 and CC6a/b; the Caco-2 cell lineexpressed CC1, CC3, CC4, CC6a,b. CC2 was not detectable in NMPpreparations of Caco2, for SW480 but was detectable in the cancer cellline CX-1. CC3, CC4 but not CC5 was detected in CX-1 NMP.

These studies demonstrate that nuclear matrix proteins isolated fromhuman colon cancer are distinct from normal adjacent and donor tissue,indicating both loss and gain from specific proteins. The presence orabsence of unique NMPs in cancer cells is useful for diagnosing diseaseand provide novel information about the function of NMPs in carcinogenesis.

C. Analysis of CC NMPs in Adenomatous Polyps of the Colon

Colon polyps (n=20) were collected through the Early Detection ResearchNetwork (EDRN) of the University of Pittsburgh Medical Center underinstitutional IRB approval. One juvenile polyp, six tubular adenoma(TA), seven tubulovillous adenoma (TVA) and six tubulovillous adenomawith high grade dysplasia (TVA with HGD) were examined. The patientsranged in age from 18 to 77 with a mean age of 58 years. 55 percent ofthe sample population was female. Diagnosis was obtained from pathologyreports, which accompanied each specimen and was confirmedhistologically. The histological partition of the colon polyps is shownin Table 3. The tissues were stored at −80° C. prior to processing.

TABLE 3 Characteristics of patients with colon polyps juvenile polyp N =1 Age = 18 Male 1 Tubular adenoma N = 6 Average age = 58 male 4; female2 Tubulovillous adenoma N = 7 Average age = 56 male 1; female 7Tubulovillous adenoma N = 6 Average age = 62 male 3; female 3 with highgrade dysplasia

The results for detecting the presence of CC2, CC3, CC4 and CC5 NMPs invarious adenomas of the colon is shown in Table 4.

TABLE 4 Presence of CC NMPs in adenomatous colonic polyps Colonicabnormality CC2 CC3 CC4 CC5 Tubulovillus adenoma 0% 83% 100% 33% withfocal high grade dysplasia (n = 6) Tubulovillus adenoma 0% 86%  86%  0%(n = 7) Tubular adenoma 0% 83% 100% 17% (n = 6) Juvenile polyps (n = 1)0%  0%  0%  0%

CC2 was not seen in any of the pre-malignant polyps. CC5 was present inonly two (33%) pre-malignant TVA with HGD and in one (17%) TA. CC3 andCC4 were present in 83%–100% in TA, in 86% in TVA and in 83%–100% in TVAwith HGD. None of the nuclear matrix proteins were seen in the juvenilepolyp, which is not a precursor of colon cancer.

CC5, which was present in only 2 pre-malignant TVA with HGD and in oneTA, but was present in all colon cancer tissues, is expressed at thejunction of an advanced adenoma and invasive colon cancer. CC5 is apromising marker for malignant potential of colon polyps because it isexpressed in the advanced polyps and in the colon cancer epithelia. CC3and CC4 were present in most adenomas, regardless of advancement. Bothproteins are expressed earlier in the development of adenomatous polypsand are also expressed in all colon cancer (Table: 2). The combinationof all four CC markers tested also provides diagnostic value for earlydetection of malignant progression in colon polyps.

Example 3 Partial Amino Acid Sequence of Colon Carcinoma NMPs

A. Method for Polypeptide Isolation and Sequencing

A partial amino acid sequence was determined for particular CC markersisolated two-dimensional gels using an adaptation of a techniquedeveloped by Gevaert et al. (35). Briefly, two-dimensional gels run withNMP preparations were negatively stained by incubating in 0.2M Imidazolefor 15 minutes, washing several times with deionized water, stainingwith a warmed solution of 0.3M zinc chloride and terminating thestaining reaction by washing in deionized water. Protein spots wereexcised from the gel and frozen at −80° C. The gel spots were thawed,pooled and stained for 20 min with 0.25% Coomassie blue in 45%methanol/9% acetic acid. The isolated gel spots were destained byagitation in destaining solution (5% methanol/7.5% acetic acid) for 1hour, washed with deionized water for 1 hour, and equilibrated in SDSpolyacrylamide sample buffer (1% SDS/10% glycerol/50 mM DTT/12 mMTris-HCl pH 7.1) for 1 hour before loading into an SDSacrylamide/agarose gel.

Following destaining, the protein from each gel spots was concentratedon a mini-agarose/acrylamide gel. The gel was formed between twopre-warmed (60° C.) glass plates (10 cm×9 cm), separated by spacers 1 cmwide and 1.5 mm thick. A strip of Whatman 3 MM paper was applied to thebottom to serve as a support for the lower agarose gel, preventing thegel from slippage during electrophoresis. A 2 cm wide×1.5 cm thickspacer was inserted between the two parallel spacers to form a samplewell for receiving the gel spots. The sample well was formed by a 2 cmwide×1.5 cm thick spacer set between two parallel spacers each 1 cmwide×1.5 cm thick inserted at the center of the glass plates andattached with adhesive tape at the top edge of the back plate.

The running gel portion of the mini-gel consisted of a 2 cm deep agarose(1.45% agarose in 0.36 M Tris-HCl pH 8.7/0.1% SDS) which was poured andallowed to set. A polyacrylamide stacking gel (5.45% acrylamide/0.13%bisacrylamide/0.12 M Tris-HCl pH 6.8/0.1% SDS) was applied over theagarose. After the stacking gel polymerized, the central well-formingspacer was removed, leaving a loading well with dimensions 2 cm high, 2cm wide and 1.5 mm thick. The mini concentration gel was then mounted ona small electrophoresis tank (BioRad, Hercules, Calif.), and the loadingslot filled with the SDS sample buffer-equilibrated gel spots. Theremaining volume was filled with blank gel pieces.

Mini-gels were run at 100 V, allowing the proteins to elute out of thecombined gel pieces and into the acrylamide. At this time, the centralspacer was re-inserted into the sample well until the dye front passedthe two parallel 1 cm wide spacers. At that point, the central spacerwas removed and electrophoresis continued until the dye front enteredthe agarose and reached the filter paper.

The agarose running gel was removed and fixed in fresh 50% methanol/10%acetic acid shaking, at room temperature for 30 minutes. The gel wasstained with 0.05% Coomassie blue stain (50% methanol/10% acetic acid)for 5 minutes and then destained in 5% methanol/7% acetic acid for 2hours with constant agitation. The protein band was then excised in aminimal volume of agarose gel, transferred to a sterile tube, anddigested thoroughly with trypsin. Trypsin fragments were subjected topreparative reverse phase C18 HPLC and fractions containing differentpeptides were isolated. An automated sequencer was used to determine asequence of fragments indifferent fractions.

B. Peptide Sequencing Results

N-terminal amino acid sequences for trypsin fragments of particularisolated CC polypeptides isolated from NMP preparations of Caco-2 cellsis shown is Table 5 while the sequences for particular CC polypeptidesisolated from NMP preparations of human colorectal cancer specimens areshown in Table 6.

TABLE 5 Partial Amino Acid Sequence of CC3 and CC6 Pro- teins Isolatedfrom Caco-2 NMP Preparation Marker No. Amino Acid Sequence (5′–3′) CC6aPXVKFNSYVDGVEV (SEQ ID NO: 1) (peak 31) EGDLIEDY (SEQ ID NO: 2) (peak17) CC3 YPVEAFN (SEQ ID NO: 3) (Peak 9) TVAPLFIVIPN (SEQ ID NO: 4) (Peak35) XVTGLTQIETLFAAPGVD (SEQ ID NO: 5) (Peak 47) SMTEAEQQQLIDDHFLFDKPVSP(SEQ ID NO: 6) (Peak 52) SLPQNIPPLTQTPV (SEQ ID NO: 7) (Peak No: 31)VLPGEIVEYSR (SEQ ID NO: 8) (Peak No: 15)

TABLE 6 Partial Amino Acid Sequence of CC Proteins Isolated from HumanColorectal Tissue NMP Preparations Marker No. Amino Acid Sequence(5′–3′) CC1 YTIFDNFLITNDEAYAEEFG (SEQ ID NO: 9) (Peak 30)QIDNPDYKGTXIHPE (SEQ ID NO: 10) (Peak 34) PAVYFKEQFLDGDGW (SEQ ID NO:11) (Peak 24) TLIVRPDNTYEVK (SEQ ID NO: 12) (Peak 18) YAVLITVLQDS (SEQID NO: 13) (Peak 13) AKTDFATFLYT (SEQ ID NO: 22) (Peak 28) CC2 NLPQE(SEQ ID NO: 14) (Peak 07) TEPELQDKIHQ (SEQ ID NO: 15) (Peak 18)TDAPSFSDIPNL (SEQ ID NO: 16) (Peak No: 20) LKYENEVALR (SEQ ID NO: 23)(Peak No: 20) XQKEDVPSE (SEQ ID NO: 17) (Peak No: 02) CC3 VYOEPLVFR (SEQID NO: 24) (Peak 31) RAPFQELYND (SEQ ID NO: 25) (Peak 31) XFYQLDAYPSGAXY(SEQ ID NO: 26) (Peak 35) CC4 VIEAFNR (SEQ ID NO: 27) (Peak 17)ILLFDYFNR (SEQ ID NO: 28) (Peak 28) VLVALEPLS (SEQ ID NO: 29) (Peak 29)CC6a NAFNDGLK (SEQ ID NO: 18) (Peak 12) YFDSFGDLSSASAIMGN (SEQ ID NO:19) (Peak 19) TYFSFHIDVSPGSAQVK (SEQ ID NO: 20) (Peak 18) CC6b SLDEQEQTK(SEQ ID NO: 21) (Peak 06)

The amino acid sequences were run against public amino acid sequencedatabases to determine if they represent previously known proteins. Thesequence of amino acids fragments generated from CC1 (SEQ ID NOS: 9, 10and 11) were consistent with the protein calreticulin, while CC1sequences with SEQ ID NO: 12 and 13 were consistent with a precursor ofcalreticulin. Calreticulin is an important multifunctionalcalcium-binding protein and is the major calcium binding protein foundin the membranes of smooth muscle sarcoplasmic reticulum and non-muscleendoplasmic reticulum (ER). Calreticulin has an immunological functionin the folding and peptide-loading of newly synthesized molecules of themajor histocompatability complex (MHC) class I protein. A complete MHCclass I molecule consists of a 3-domain alpha-chain and a smallerβ2-microglobulin bound to a short peptide fragment. When theβ2-microglobulin first binds to the alpha-chain, this partially foldedheterodimer binds to a complex of proteins including calreticulin.Calreticulin's binding of the MHC molecule is regulated by glucosetrimming of nascent N-linked oligosaccharides—the oligosaccharide moietyin the alphal domain and a residue within the alpha3 domain of the MHCclass I molecule are critical for intersection with calreticulin. Afterpeptide loading and deglucosylation of N-linked glycans, calreticulindissociates from the heterodimer.

Example 4 Identification of Calreticulin in Colon Cancer NMPPreparations

The presence of calreticulin in colon cancer was evaluated using NMPpreparations as well as nuclear and cytoplasmic extracts.

A. Methods

Nuclear and Cytoplasmic Extraction: NE-PER nuclear and cytoplasmicextraction reagents (Pierce Chemical Co., Rockford, Ill.) were used forthe preparation of nuclear and cytoplasmic extracts. The proteinconcentration was quantitated by Coomassie Plus protein assay (PierceChemical Co., Rockford, Ill.) with bovine serum albumin as a standard.

One-Dimensional Immunoblot: One-dimensional immunoblot analysis wasperformed according to standard established protocols. Ten μg of eachsample of extracted NMPs was suspend in PBS (phosphatase buffered saltsolution) and nuclear and cytoplasmic extracts suspend in nuclearextraction reagent (NER) or in cytoplasmic extraction reagent (CERII)were separated by 12% SDS-PAGE. Ten μl of Rainbow markers (Amersham LifeSciences, Arlington Heights, Ill.) were also loaded. Proteins were thentransferred to a polyvinylidene difluoride membrane (Millipore, Bedford,Mass.), and the membrane was blocked overnight in 4% nonfat dry milk inPBS with 0.2% Tween® 20 at 4° C. The membrane was then washed with PBSand 0.2% Tween® 20, followed by a 1-h incubation with a 1:2000 dilutionof anti-calreticulin IgG (Research Diagnostics, N.C., Flanders, N.J.)and 2% nonfat dry milk with 0.2% Tween® 20 in PBS. The membrane wasfurther washed with PBS and 0.2% Tween® 20 and incubated for 1 h in a1:5000 dilution of goat anti-rabbit IgG (Amersham Life Sciences,Arlington Heights, Ill.) secondary antibody conjugated with horseradishperoxidase (Amersham Life Sciences, Arlington Heights, Ill.). Fordetermination of the relative purity of each protein extraction, themembranes were probed with a monoclonal α-tubulin mouse antibody (1:500)(specific for cytoplasmic tubulin) and 2% nonfat dry milk with 0.2%Tween® 20 in PBS. These membranes were further washed with PBS and 0.2%Tween®20 and incubated for 1 hour in a 1:5000 dilution of goatanti-mouse IgG (Amersham Life Sciences, Arlington Heights, Ill.)conjugated with horseradish peroxidase. The membranes were washed againwith PBS and 0.2% Tween® 20 and the proteins detected bychemiluminescence reaction using the ECL immunoblot kit (Amersham LifeSciences, Arlington Heights, Ill.).

Two-dimensional immunoblot: After performing 2-D electrophoresis, thearea of the gel where the spot was located and which the peptidesequence resulted in identification as calreticulin was removed and wastransferred to a polyvinylidene difluoride membrane (Millipore, Bedford,Mass.). This area of the gel was utilized instead of the entire gel,because the large gel format makes blotting and processing difficult.Thereafter, the same procedure described above for the one-dimensionalimmunoblot was followed by a 1-h incubation with a 1:2000 dilution ofanti-calreticulin IgG (Research Diagnostics, Inc., Flanders, N.J.) with2% nonfat dry milk with 0.2% Tween® 20 in PBS. The membrane was furtherwashed with PBS and 0.2% Tween® 20 and incubated for 1 h in a 1:5000dilution of goat anti-rabbit IgG (Amersham Life Sciences, ArlingtonHeights, Ill.) secondary antibody conjugated with horseradish peroxidase(Amersham Life Sciences, Arlington Heights, Ill.). The membrane waswashed again with PBS and 0.2% Tween® 20 and proteins were detected by achemiluminescence reaction using the ECL immunoblot kit (Amersham LifeSciences, Arlington Heights, Ill.).

B. Results

The two-dimensional immunoblot of CC1 probed positive with theanti-calreticulin antibody. A one-dimensional immunoblot of coloncancer, normal adjacent and normal donor colon tissue cytoplasmic,nuclear and nuclear matrix protein fractions was probed with antibodiesspecific for calreticulin and α-tubulin (cytoplasmic 51 kD protein).Tubulin was not detectable in nuclear and the nuclear matrix proteinfractions indicating the that lack of contamination of cytoplasmicmaterial in these fractions. Calreticulin was detected (about a 60 kDband) in human colon cancer tissue, adjacent normal tissue and normaldonor tissue in all three different protein fractions. These resultsshow that calreticulin is present in the nuclear protein fraction andmore specifically in the nuclear matrix protein fraction of colon cancercells, and that such presence is not due to contamination by cytoplasmiccalreticulin. Calreticulin was less detectable in normal adjacent andnormal donor colon tissue NMP preparations by one dimensionalinmunoblotting.

Example 5 Identification of NMPs Characteristic of Liver Metastases andPrimary Colon Cancer Cells

A. Expression of L1–L5 Proteins in NMPs from Various Sources

High-resolution two-dimensional gel electrophoresis of various NMPpreparations identified five proteins (L1–L5) of clinical value (Table7). NMPs L1–L5 were detectable in about 83% to 100% of 2D gels for NMPpreparations of the various colon cancer to liver metastases. L1–L5 wereundetectable in NMP preparations of normal hepatocytes. Three proteins,L1, L2 and L5, were undetectable in NMP preparations of normal donorliver tissue, and two proteins, L3 and L4, were detectable in a lowerpercentage of gels prepared from NMP preparations of normal donor livertissue as compared to that of colon to liver metastasis. Four of theproteins (L1, L2, L4 and L5) were detectable in less than 40% in theadjacent normal liver tissue samples (Table 7), while protein L3 wasdetectable in 92% of the adjacent liver tissue samples (Table 7).

Proteins L1, L2, L3 and L4 also were detectable in 60% or more of gelsprepared using NMP preparations of primary colon cancer cells (Table 7).Protein L5, on the other hand, was detectable in only 20% of gelsprepared using NMP preparations of in primary colon cancer cells (Table7). Proteins L3 and L4 were expressed in 100% of gels prepared using NMPpreparations of the normal adjacent colon and 100% and 75% respectivelyfor donor colon tissue. Protein L2 was expressed in 10% of the adjacentnormal colon tissue, while L1 was not detectable in the adjacent normalcolon, but was detectable in one of the four donor colon samples (25% inTable 7). Electrophoretic characteristics of the identified proteins areshown in Table 7.

Thus, L1–L5 are differentially expressed in normal liver metastasesversus normal hepatocytes. L2 and L2 are the most differential of allproteins, the difference being seen in both colon liver metastasesversus adjacent liver tissue and in primary colon cancer versus adjacentcolon tissue. L3 is differential for liver metastases versus adjacentliver tissue or for primary colon cancer versus normal adjacent colon.

TABLE 7 Characteristics of NMPs in Liver Metastases and Other Liver andColon Tissues 2D Gel Tissue Expression Colon Ca. to Normal Normal NormalNormal Liver Adj. donor Colon Adj. donor Markers/ Mr. mets. liver liverNormal Ca. Colon colon NMPs (kD) PI (n = 12) (n = 12) (n = 3)Hepatocytes (n = 10) (n = 10) (n = 4) L₁ 50 6.01  92% 42% 0% 0% 70%  0% 25 L₂ 20 5.73 100% 17% 0% 0% 100%   10% 0% L₃ 17 6.09 100% 92% 33%  0%90% 100% 100 L₄ 17 6.00 100% 17% 33%  0% 60% 100% 75%  L₅ 18 5.88  83% 8% 0% 0% 20%  0% 0% Adj. = adjacent; Ca—cancer; mets = metastasisB. Analysis of NMPs from Liver and Colon Cancer Tissue and Cell Lines

Tissue samples are complex mixtures of epithelial, stromal,immunological and other cell types. To determine whether the nuclearmatrix changes detected actually represented changes that were occurringin the neoplastic cells, as well as to identify potential models, theNMP composition of two human primary liver cancer cell lines wereexamined. While the NMP fingerprints from the pure cell lines would beexpected to be distinct from the three dimensional complex of livermetastasis and colon cancer specimens they serve as tools for generatingreagents as well as examining a single cell type. The human liver cancercell lines Huh 7 and HepG 2 and the human colon cancer cell lines Caco-2and SW480 were grown and their NMPs were isolated. These NMPs were thenseparated and analyzed as described above. The cell line HepG 2expressed none of the liver metastasis associated proteins, and the Huh7 cell line expressed one of the liver metastasis associated proteins(L1). The cell line Caco-2 expressed two of the proteins (L3, L2) andthe cell line SW480 expressed just one protein (L2). Electrophoreticcharacteristics of the identified proteins in these cell lines are shownin Table 8.

TABLE 8 Characteristics of NMPs in Colon Cancer Cell Lines Markers/ Mr.2D-Gel Cell Expression NMPs (kD) PI Hep G₂ Huh 7 Caco-2 SW 480 L₁ 506.01 0% 0% 0% 0% L₂ 20 5.73 0% 0% 100%  100%  L₃ 17 6.09 0% 0% 100%  0%L₄ 17 6.00 0% 0% 0% 0% L₅ 19 5.88 0% 0% 0% 0%

These studies demonstrate that nuclear matrix proteins uniformly presentin NMP preparations from human liver metastasis are not readily presentin NMP preparations of normal donor tissue or isolated hepatocytes. Inaddition, in some cases, the normal adjacent liver tissue containsalterations in the nuclear matrix pattern similar to those found inliver metastasis. These five proteins were also found to varying degreesin primary colon cancer, but were not found in other cancer types.

The functional identification of these proteins and their detectionthrough the generation of NMP antibodies could be used to develop testsfor colon cancer prognosis and early detection of metastases. Antibodiesare generated to detect specific nuclear matrix proteins in the blood ortissue samples. Assay is developed that include the detection andcombination of individuals proteins identified herewith. Development ofassays with these antibodies potentially serve as tumor marker with highsensitivity and specificity.

Additionally the presence of unique NMPs in liver metastasis or the upregulation in cancer cells could provide novel information about theirfunction in development of metastasis and provide us with additionaltargets for anticancer therapies.

Example 6 Isolation of “L” Proteins from 2-D Gels and Sequencing

Methods for isolating and sequencing L proteins were as described forthe CC proteins in Example 3. N-terminal amino acid sequences fortrypsin fragments of particular isolated L polypeptides isolated fromNMP preparations of colorectal metastases to liver cells is shown isTable 9.

TABLE 9 Partial Amino Acid Sequence of L2 and L5 Pro- teins Isolatedfrom Human Colorectal Metastases to Liver NMP Preparations Marker No.Amino Acid Sequence (5′–3′) L2 QFYQLDAYPSGAWYYVP (Peak *) (SEQ ID NO:30) FFVALFPEVF (Peak 23) (SEQ ID NO: 31) QFYQLDAYPSGAWYYVP (Peak 31)(SEQ ID NO: 32) L5 TDAPSFSDIPNL (Peak No: 20) (SEQ ID NO: 33)FFVALFPEVFGK (Peak No: 26) (SEQ ID NO: 34) QFYQL (Peak No: 28) (SEQ IDNO: 35) AVPYPQRDMPI (Peak 38) (SEQ ID NO: 36)

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The invention thus has been disclosed broadly and illustrated inreference to representative embodiments described above. Those skilledin the art will recognize that various modifications can be made to thepresent invention without departing from the spirit and scope thereof.

1. A monoclonal antibody which binds to a peptide consisting of SEQ IDNO: 28.